© 1997 American Heart Association, Inc.
Articles |
Fas Is Expressed in Human Atherosclerotic Intima and Promotes Apoptosis of Cytokine-Primed Human Vascular Smooth Muscle Cells
From the Vascular Medicine and Atherosclerosis Unit, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Mass.
Correspondence to Yong-Jian Geng, MD, PhD, Vascular Medicine and Atherosclerosis Unit, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 221 Longwood Avenue, Boston, MA 02115.
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Abstract |
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Abstract The membrane protein Fas/Apo-1/CD95 signals programmed cell death or apoptosis in activated T lymphocytes. Vascular smooth muscle cells (SMCs) bear markers of programmed cell death or apoptosis in advanced atherosclerotic plaques that contain immune cells eg, macrophages and T lymphocytes. This study tested the hypothesis that the Fas death-signaling pathway contributes to apoptosis of SMCs exposed to proinflammatory cytokines produced by these immune cells during atherogenesis. All atherosclerotic plaques examined (n=14) contained immunoreactive Fas. The majority of the Fas+ SMCs localized in the intima of the plaques, whereas the medial SMCs expressed Fas antigen less prominently. Double staining for DNA fragments (TUNEL) and Fas or cell identification markers colocalized Fas with TUNEL+ SMCs in the areas that contained CD3+ T cells and CD68+ macrophages, suggesting a role for Fas in the induction of SMC apoptosis by activated T cells during atherogenesis. In culture, stimulation with interferon-
, tumor necrosis factor-
, and interleukin-1ß
increased expression of Fas in SMCs. Incubation with an activating
anti-Fas antibody triggered apoptosis of the
cytokine-primed but not the untreated SMCs, as demonstrated by
TUNEL and electrophoresis of oligonucleosomal DNA fragments. These data
suggest that activation of the Fas death-signaling pathway contributes
to the induction of SMC apoptosis during atherogenesis and
furnish a mechanism whereby immune cells and their
cytokines promote this cell death process related to vascular
remodeling and plaque rupture.
Key Words: atherosclerosis • smooth muscle cells • CD95 • T cells • cytokines • apoptosis
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Introduction |
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Apoptosis, a form of genetically programmed cell death, represents a major mechanism by which tissues eliminate unwanted or harmful cells and maintain homeostasis.1 Rapidly accumulating evidence indicates the involvement of deregulated apoptosis in the pathogenesis of various diseases including atherosclerosis. Recent work by this2 and other laboratories3-6 demonstrates the presence of numerous cells that bear markers of apoptosis in advanced human atherosclerotic plaques as well as in experimental models of atherosclerosis5,7 and restenosis.8 Imbalance between cell death and survival may substantially affect the cellularity and integrity of the blood vessel wall and contributes to the pathogenesis of atherosclerosis.
Atherosclerotic plaques contain different types of immune cells,
particularly macrophages and T
lymphocytes.9 The existence of activated
macrophages and T lymphocytes in various stages of
atherosclerosis points to a role for these immune cells
in the regulation of proliferation, the differentiation, and the death
of vascular cells in atherosclerotic lesions. The proinflammatory
cytokines, interferon- (IFN-),10 a
product of activated T lymphocytes, and tumor necrosis
factor (TNF) and interleukin-1 (IL-1),11 two
cytokines elaborated by activated macrophages,
found in atherosclerotic plaques can profoundly alter functions of
vascular smooth muscle cells (SMCs). For example,
IFN-12,13 stimulates the expression of major
histocompatibility complex class II antigen but inhibits growth of
vascular SMCs. TNF-14 or
IL-115 can stimulate SMC proliferation and
enhance IFN–-induced expression of major histocompatibility complex
class II antigen. Simultaneous exposure to these three
proinflammatory cytokines promotes apoptosis of
cultured vascular SMCs in a concentration- and time-dependent
fashion.16 These observations imply that in
atheromatous plaques, activated immune cells
may trigger apoptosis of SMCs by producing these
cytokines.
Several gene products involved in regulation of apoptosis
may mediate apoptosis of SMCs including c-myc,
p53, and Bcl-2.17,18
Fas/Apo-1/CD95, a novel member of the nerve growth factor/TNF receptor
gene family, mediates apoptosis of target cells attacked by
activated cytotoxic T cells.19 In
addition to the cells of the immune system, other cell types, including
those in the liver, the lung, and the heart, also express this molecule
constitutively.20,21 Stimulation with the
cytokines IFN- and TNF can augment expression of Fas and
thereby enhance the apoptotic effect of Fas in certain cell
types.22-24 The death pathway triggered by
activation of Fas involves a group of proteases related to
interleukin-1ß converting enzyme (ICE),25 a
cysteine protease found in a variety of normal and diseased tissues
including atherosclerotic plaques.2 Activation of
Fas requires binding by Fas ligand (FasL) and can be mimicked in vitro
by certain anti-Fas antibodies. cDNA cloning identified FasL as a novel
member of the TNF family and a primary cytotoxic factor produced by
cytotoxic CD8+ T
lymphocyes.26,27 Because atheroma
contains lymphocytes including both CD4+ helper T
cells and CD8+ cytotoxic T cells as well as
antigen-presenting cells including macrophages, a local
immune response may employ the Fas/FasL death pathway to destroy
vascular target cells during atherogenesis.
This study tested the hypothesis that the Fas/FasL death-signaling pathway may contribute to apoptosis of vascular SMCs exposed to the cytokines derived from activated macrophages and T cells during atherogenesis. We examined expression of Fas in human atherosclerotic lesions and in human SMC cultures stimulated with the proinflammatory cytokines. We also investigated induction of apoptosis in the cytokine-primed SMCs by activation of the Fas death-signaling pathway with anti-Fas antibody. This study sheds new insight into the mechanism by which vascular SMCs undergo apoptosis in atherosclerotic lesions and points to the role for the Fas death-signaling pathway in regulation of SMC death during atherogenesis.
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Methods |
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Reagents
Recombinant human IFN-
was purchased from Genzyme Inc, and
human TNF- and IL-1ß were purchased from Endogen Inc. Two mouse
monoclonal antibodies against human Fas (UB2 IgG and CH-11 IgM) were
supplied by Medical and Biological Laboratories Co. Monoclonal
antibodies against SM -actin, the macrophage marker CD68,
and the T-cell antigen CD3 were purchased from DAKO Inc. The nucleic
acid-binding fluorochromes acridine orange and ethidium bromide were
purchased from Sigma.
Atherosclerotic Plaques
Human carotid atherosclerotic plaques were obtained from
patients undergoing carotid endarterectomy. The
arterial specimens were immersed in ice-cold Hanks'
solution immediately after removal and then washed and fixed in 10%
formalin for preparation of paraffin sections. For preparation of
cryostat sections, the tissue was immersed in optimal cutting
temperature tissue processing medium (O.C.T., Miles
Diagnostics), snap-frozen in liquid nitrogen, and stored at
-80°C. The study of normally discarded human tissues was approved by
the Institutional Human Investigation Review Committee.
Isolation and Culture of SMCs
Human vascular SMCs were isolated from the tunica media of aorta
and cultured in DMEM (GIBCO) supplemented with 10% fetal calf serum
and antibiotics.28 The cells were identified as
vascular SMCs by their characteristic growth pattern in "hills and
valleys" and by immunofluorescence with anti–SM
-actin monoclonal antibody. They were passaged by trypsinization,
plated at a density of 2x104 cells per
milliliter, and used for experiments at two to seven passages.
Stimulation of SMCs With Cytokines and Anti-Fas
Antibody
Human SMCs at subconfluent density were treated with
cytokines IFN- (500 U/mL), IL-1ß (100 U/mL), and TNF-
(500 U/mL), alone or together, for 24 or 48 hours. After treatment with
the cytokines, cells were washed three times with PBS and then
incubated in serum-free medium with or without the mouse anti-human Fas
monoclonal antibody CH-11 at 200 or 500 ng/mL for up to 1 week.
At the end of the incubation with the cytokines and the
antibody, the cells were analyzed for apoptosis and Fas
expression, as described next.
Cell Viability
The viability of vascular SMCs was determined by staining with
the nucleic acid-binding fluorochromes acridine orange and ethidium
bromide.16 SMCs (104/0.5
milliliter per chamber) were cultured in eight-chamber slides (Nunc).
The cells pretreated with IFN-, TNF-, and/or IL-1ß for 24 hours
were incubated with anti-Fas antibody. After this incubation, the
chamber slides were incubated on ice with the DNA binding dyes,
acridine orange and ethidium bromide, at 10 µg/mL each. After
staining for 2 minutes, the slides were covered and observed under a
fluorescent microscope. Viable (green fluorescent
nuclei) and nonviable (red or orange fluorescent nuclei) cells
were counted. For each sample, at least 200 cells were counted in
different high-power fields. The percentage of viable cells was
determined by the following formula: % cell viability=100x(number of
viable cells)/(total number of cells).
In Situ 3' End Labeling of DNA Fragments (Terminal Transferase End
Labeling [TUNEL])
In situ labeling of DNA fragments has been widely used as a
biochemical marker of apoptosis in
vivo.29,30 We performed in situ labeling of DNA
fragments by use of terminal deoxyribonucleotide
transferase-mediated dUTP nick end labeling (TUNEL) based on an ApoTag
in situ apoptosis detection Kit (Oncor Inc) in atherosclerotic
lesions and SMC cultures. TUNEL was carried out in paraffin sections of
atherosclerotic plaques and normal control vessels because of (1)
better preservation of the morphology and (2) lower activities of
endogenous nucleases in the tissues embedded in paraffin.
Briefly, the paraffin was removed from the sections by immersing in
xylene, rehydrated in 100%, 95%, 75%, and 0% ethanol, and incubated
in PBS with 2% H2O2 to
inactivate endogenous peroxidases. For
detection of DNA fragments in cultured cells, SMCs grown on a chamber
slide were washed and fixed in 4% formaldehyde in PBS. After
incubation with proteinase K (20 µg/mL) for 20 minutes, DNA
fragments were labeled with digoxigenin-conjugated dUTP and the
terminal transferase for 1 hour. The incorporation of digoxigenin dUTP
into DNA was determined by incubating the sections with
peroxidase-conjugated antibody against digoxigenin at room temperature
for 30 minutes. The chromogenic substance DAB was used as
the peroxidase substrate to visualize the staining. For TUNEL of
cultured SMCs, we cultured the cells in eight-chamber slides and
treated the cells with or without cytokines and then with
anti-Fas antibody. Cells were washed in PBS, fixed, incubated with
proteinase K, and then labeled with digoxigenin-conjugated dUTP and the
enzyme TdT. After incubation with the peroxidase substrate DAB, the
slides were washed in PBS, counterstained in 0.5% methyl green in 0.1
mol/L sodium acetate solution (pH 4.0) for 5 minutes, and
mounted in Permount medium for microscopic observation.
TUNEL+ nuclei were identified by brown nuclear
stain and altered nuclear morphology such as chromatin condensation and
margination. Nonspecific staining of TUNEL was reported in
atherosclerotic plaques.5 To ascertain the
specificity of TUNEL, control staining was performed by omitting TdT or
treating slides with EDTA at 10 mmol/L to eliminate free
calcium and to inhibit endogenous DNase activity. Four
hundred cells were counted in a high-power field. The percentage of
TUNEL+ cells was calculated by dividing the
number of TUNEL+ cells by the total number of
cells.
Immunohistochemistry
Sections of atherosclerotic plaques and normal vessels were used
for immunohistochemistry with mouse monoclonal antibodies against SM
-actin, T-cell CD3, and Fas. After fixing in acetone for 10 minutes
at -20°C, sections were incubated with 1:50 normal horse serum for
30 minutes at room temperature. Sections were washed in PBS and then
incubated with each antibody diluted at 1:100 in PBS for 1 hour. After
incubation and washing again, the slides were incubated with
biotin-conjugated second antibodies at dilution of 1:200. Biotinylated
rabbit anti-mouse IgG (Vector Laboratories Inc) was used for detecting
the stains with these monoclonal antibodies. An avidin-alkaline
phosphatase-substrate system (Vectastain ABC kit, Vector) was used for
the visualization of the immunostains. In some experiments,
we performed double staining with a combination of TUNEL and
immunohistochemistry with the antibodies to these cellular antigens.
Sections were first stained by TUNEL for visualizing DNA fragmentation
and then by immunohistochemistry for determination of cell antigens.
Enumeration of positively immunostained cells was performed
by counting cells reacted with antibody in a high-power field. Four
hundred cells were counted for each sample independently by two
persons. The percentage of positive cells was calculated by dividing
the number of positive cells by the total number of cells.
Flow Cytometry
SMCs treated with or without cytokines in six-well
plates were washed and then incubated with PBS containing 5
mmol/L EDTA on ice for 5 minutes. The cells were collected by
gently scraping into a microfuge tube and centrifuged at
3000xg for 2 minutes. Cell pellets were suspended in 100
µL of PBS with anti-Fas antibody (1:100) and incubated on ice for 30
minutes. After incubation, the cells were washed again and incubated
with second anti-mouse IgG conjugated with FITC (1:200) for 20 minutes.
The cells were suspended in 500 µL of PBS containing 25
µg/mL propidium iodide. Fluorescence-activated
cell sorter (FACS) analysis of the cells was performed with the
use of the flow cytometer FACSort. At least 5000 cells were counted,
and data were evaluated by using the FACS Cellquest program.
Immunoblotting Assay
Cells cultured in 80-cm2 flasks were
washed with ice-cold PBS three times and then collected into a 15-mL
centrifugation tube. After
centrifugation at 2000 revolutions per minute, cell
pellets were suspended in 50 mmol/L Tris-HCl buffer
containing 10 mmol/L EDTA, 10 mmol/L PMSF, and
0.1% Triton 100. The cell lysate was transferred to a microfuge tube
and centrifuged at 3000xg for 5 minutes to remove
nuclei. The protein content in the supernatant was determined by
Bradford's method. Thirty micrograms of protein was loaded into 10%
SDS-PAGE under reducing conditions. After electrophoresis, the protein
was transblotted onto a PVDF membrane (Millipore Inc) in 20
mmol/L Tris-glycine buffer containing 10% methanol. The
membrane was incubated in PBS blocking buffer with 0.02% Tween 20 and
3% fat-free milk for 30 minutes and then anti-Fas antibody (1:2000) in
the blocking buffer for 1 hour. After incubation with the primary
antibody, the membrane was washed in PBS with 0.02% Tween 20 three
times, incubated with alkaline-phosphatase-conjugated rabbit anti-mouse
IgG (1:5000), and developed in the substrate solution of 20
mmol/L Tris-HCl, 0.4 mg/mL naphthol AS-Mx phosphate and 1
mg/mL Fast Red TR salt (Sigma).
RNA Isolation and Reverse Transcriptase–Polymerase Chain
Reaction (RT-PCR)
Total RNA was isolated from SMCs using the method
presented by Chomczynski and Sacchi31
with modification. Briefly, cells were cultured in
80-cm2 flasks and stimulated with or without
cytokines. After stimulation, the culture media were removed,
and 1 mL of lysis buffer containing 4 mol/L guanidinium
isothiocyanate, 10 µM 2-mercaptoethanol, 25 mmol/L sodium
citrate (pH 7.0), and 0.5% N-lauroylsarcosine was added,
followed by the addition of 0.1 volume sodium acetate (2 mol/L,
pH4.0) and mixing with phenol and chloroform-isoamyl alcohol 49:1 (0.2
volume). The mixture was centrifuged at 10 000xg
at 4°C for 10 minutes. RNA at the upper phase was collected and
precipitated in isopropanol (vol/vol, 1:1) at -80°C over
night. RNA pelleted by centrifugation was washed and
dissolved in TE buffer. Two hundred fifty nanograms of total RNA was
reverse-transcribed into cDNA with 2.5 U/mL of Moloney murine leukemia
virus reverse transcriptase (Perkin Elmer) at 42°C for 30 minutes in
20 µL RT buffer (50 mmol/L random hexamers, 1
mmol/L of each dNTP, 1 U/mL RNase inhibitor, and
2.5 mmol/L MgCl). After denaturation at 94°C for 2
minutes, cDNA of Fas was amplified by PCR with a set of primers
specific for human Fas. The reaction of PCR was composed of 20 µM
each of 5' and 3' primers and 2.5 U Taq DNA polymerase (AmpliTaq,
Perkin Elmer-Cetus) and run for 30 cycles. Products of PCR were
analyzed by agarose gel electrophoresis and visualized by
ethidium bromide staining. In some experiments, the RT-step was omitted
or the genomic DNA was used to replace cDNA templates from RNA to
determine the specificity of PCR amplification for Fas mRNA.
DNA Isolation and Electrophoresis DNA Fragmentation
Analysis
Cells (5x106) were lysed in 1 mL of DNA
extraction solution containing 20 mmol/L Tris-HCl (pH 7.4),
0.1 mol/L NaCl, 5 mmol/L EDTA, and 0.5% SDS. The
lysates were incubated with 100 µg/mL proteinase K at 37°C
for 16 hours. After incubation, 1 mL of phenol/chloroform (1:1)
was mixed well with the enzyme-digested cell lysates, and the mixture
was then centrifuged at 10 000g for 20 minutes; DNA
in the upper (aqueous phase) was incubated with 5 µg/mL
DNase-free RNase A at 37°C for 1 hour and extracted with
phenol/chloroform again. DNA was collected by precipitation with
1 mL isopropanol and 0.1 mL 5 mol/L NaCl at -20°C overnight.
After centrifugation, the resulting DNA pellets were
washed with 75% ethanol and air-dried. DNA was dissolved in 10
mmol/L Tris-HCl and 1 mmol/L EDTA, and its
concentration was determined at 260 nm by spectrophotometry. DNA
electrophoresis was carried out in 1.5% agarose gels containing 1
µg/mL ethidium bromide, and DNA fragments were visualized by
exposing the gel to ultraviolet light.
Statistical Analysis
The difference between means was evaluated using Student's
t test. For statistic analysis of data from multiple
groups, we used ANOVA. Significant levels were established when
P values were less than 0.05.
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Expression of Fas Antigen in the Intima of Human Atherosclerotic Plaques
Fas was initially identified as a surface protein on activated T lymphocytes.19 We examined whether cells in advanced human atherosclerotic plaques express this death-signaling molecule by immunohistochemical study of sections of carotid atheroma (n=14). All sections examined in this study exhibited some degrees of anti-Fas immunostaining throughout the lesions (Fig 1
and Table 1). In the lipid core area, 78% of cells
with intact nuclei were positively stained with anti-Fas (Table 1).
Approximately 46% of cells in the shoulder of plaques stained
positively for Fas (Table 1). Interestingly, nearly 67% of cells were
Fas+ in the fibrous cap (Fig 1a and Table 1), a
region overlying the lipid core of plaques and prone to rupture. Fas
localized predominantly in the cytoplasm of intimal cells in
atherosclerotic arteries (Fig 1b). In contrast, cells in the underlying
media exhibited moderate anti-Fas immunoreactivity (Fig 1a). An
irrelevant mouse IgG showed no staining in atheromatous
arteries (Fig 1f).
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The two major cellular components of the plaques, macrophages
and T lymphocytes, can express Fas. As expected, in the areas with the
Fas immunostain, we observed both
CD68+ and CD3+ cells (Fig 1c
and 1e). However, lesions contained more Fas+
cells (62%) than CD68+ macrophages
(13%) or CD3+ T lymphocytes (9.5%), suggesting
that in addition to these immune cells, other cell types might produce
Fas antigen. Indeed, 65% of the intimal cells in lesions stained for
-actin, indicating that they were SMCs (Fig 1). The actin-positive
cells colocalized with the Fas antigen (Fig 1d), establishing the
presence of Fas-expressing SMCs in the plaques.
Colocalization of Fas Antigen With Vascular SMCs Bearing Markers of
Apoptosis in Atherosclerotic Plaques
In situ detection of DNA fragments using the TUNEL technique
supports the occurrence of apoptosis in human atherosclerotic
lesions. To explore possible involvement of Fas in induction of
apoptosis in atherosclerotic plaques, we performed double
staining with a combination of TUNEL and immunohistochemistry with
anti-Fas antibody. TUNEL+ cells in the plaques
often expressed anti-Fas immunoreactivity (Fig 2). These cells showed morphologic
changes such as chromatin condensation and nuclear fragmentation
characteristic of apoptosis (Fig 2b). Quantification of
double-stained cells showed that 20% Fas+ cells
exhibited TUNEL positivity. Some TUNEL+ cells
reacted with anti–SM -actin antibody (Fig 2d), indicating their
identity as SMCs. CD3+ T lymphocytes also
localized in this region with TUNEL+ SMCs (Fig 2c). The presence of Fas+ SMCs bearing markers of
apoptosis in atherosclerotic plaques containing T lymphocytes
suggests that the Fas death-signaling pathway might mediate
apoptosis of vascular SMCs triggered by activated T
lymphocytes.
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Enhancement of Expression of Fas in Vascular SMCs Exposed to
Proinflammatory Cytokines
The above finding that Fas coexisted with
TUNEL+ SMCs in the plaques raised the possibility
that products of activated immune cells may regulate
expression of Fas in the plaques. To test this possibility, we
analyzed Fas expression by human SMCs treated with or without
cytokines. Cultured human SMCs constitutively expressed Fas
antigen (Fig 3a). TNF- at 500 U/mL did
not change expression of Fas (Fig 3b). However, stimulation with
IFN- at 500 U/mL for 24 hours increased the intensity of the
anti-Fas immunostain (Fig 3c). Combining IFN- with
TNF- (both cytokines found in atheroma) also
yielded Fas expression (Fig 3d). These data establish that
cytokines can regulate surface expression of Fas in cultured
human SMCs.
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To quantify expression of Fas on the surface of
cytokine-stimulated and unstimulated SMCs, we performed
fluorescent flow cytometry with the same anti-Fas antibody. In
a control culture 33% of SMCs showed positive anti-Fas stain (Fig 4a, gated in the region, M1). IFN- at
500 U/mL markedly increased the number of Fas+
cells in the culture (Fig 4b) but only slightly increased mean
fluorescent intensity (Table 2).
By contrast, TNF- and IL-1ß alone induced little Fas expression
(Fig 4c and 4d and Table 2). Simultaneous treatment with
IFN- and the other two cytokines not only increased the
number of Fas+ cells but also the mean intensity
of anti-Fas immunostaining per cell (Fig 4e and 4f and
Table 2).
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Consistent with the data of immunohistochemistry shown above,
immunoblotting with anti-Fas visualized a protein band
at about 42 kDa, corresponding to the molecular weight of the
membrane-bound form of Fas, in SMCs either basally or at higher levels
after cytokine stimulation (Fig 5). Simultaneous stimulation
with IFN- (500 U/mL), TNF- (500 U/mL), and IL-1ß (100 U/mL)
increased immunoreactive Fas protein (Fig 5). To determine if
stimulation with a combination of these cytokines affects
general synthesis of cellular proteins, we analyzed the protein
extract of SMCs by SDS-PAGE. Staining the gels with the protein-binding
dye Coomassie brilliant blue G250 illustrated a similar pattern of
total cellular proteins fractionated in SDS-PAGE between
cytokine-treated and untreated cells (Fig 5, lower panel),
suggesting that the cytokine stimulation did not alter overall
synthesis of cellular proteins.
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To demonstrate the expression of Fas mRNA in SMCs, we performed RT-PCR
of Fas mRNA with a set of primers designed from Fas
cDNA.32 PCR amplification detected a fragment of
Fas cDNA at the expected size 448 bp in both control and
cytokine-treated SMCs (Fig 6),
consistent with the above results from
immunoblotting assay.
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Induction of Apoptosis of SMCs Primed With
Cytokines by Activation of Fas With Anti-Fas Antibody
To determine the function of Fas expressed in human vascular SMCs,
we used a mouse monoclonal anti-Fas IgM (CH-11) to activate the
Fas signaling pathway and induce apoptosis in this cell type.
This antibody reportedly induces apoptosis in many other types
of Fas-expressing cells.24,33,34 Treatment with
CH-11 at 200 ng/mL for a period up to 1 week in DMEM with or
without serum did not change cell viability. However, in the cells
pretreated with the cytokines IFN-, TNF-, and IL-1ß,
incubation with anti-Fas antibody in serum-free media significantly
reduced cell viability (Fig 7).
Pretreating the cells with a combination of the two or three
cytokines markedly enhanced the cytotoxic effect of anti-Fas
antibody (Fig 7). The death induced by this anti-Fas antibody depended
on the concentration of IFN-. The maximum reduction in the viability
of SMCs occurred in the cells pretreated with this cytokine at
500 U/mL (Fig 8).
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The cytokine-primed, CH-11–stimulated SMCs showed morphologic
alterations typical of apoptosis including cell shrinkage,
blebbing, and fragmenting into apoptotic bodies (Fig 8). End
labeling of DNA fragments by TUNEL revealed that after priming with
IFN- (500 U/mL) and IL-1ß (100 U/mL), numerous
TUNEL+ cells were found in the SMC cultures
exposed to the anti-Fas antibody for 6 days (Fig 8). By contrast, under
the same conditions, the cytokine-untreated control cultures
contained few or no TUNEL+ cells (Fig 8a),
suggesting the requirement of priming with the cytokines for
induction of SMC death by ligation of Fas.
To obtain further biochemical evidence for apoptosis of SMCs
stimulated with cytokines and anti-Fas antibody, we isolated
genomic DNA and analyzed the size of DNA fragments by agarose
gel electrophoresis. After electrophoresis, we observed
oligonucleosomal DNA fragments at about 180 bp or multiples in SMCs
pretreated with cytokines but not in untreated control cells
(Fig 9). Pretreatment with either IFN-
or TNF- alone increased the anti-Fas antibody-induced fragmentation
of SMC DNA only moderately. However, simultaneous
application of both cytokines produced a marked increase in DNA
fragmentation after stimulation with anti-Fas antibody at 200
ng/mL for 3 days. The addition of IL-1 in the pretreatment
further enhanced anti-Fas antibody cytotoxicity toward SMCs (Fig 9).
Thus, stimulation with the anti-Fas antibody CH-11 promoted the DNA
fragmentation of the SMCs primed with cytokines but not in
untreated control cells.
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Discussion |
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The Fas/Apo-1/CD95 death-signaling pathway plays an important role in activation-induced apoptotic cell death.19 This study offers evidence for the involvement of this death pathway in apoptosis of vascular SMCs in human atherosclerotic plaques and in culture. Although cells of the normal arterial wall can express Fas at a basal level, increased expression of Fas occurs in the intima of atherosclerotic arteries, a region containing numerous activated macrophages and T lymphocytes.35,10 The activated immune cells may release cytokines, shown here to enhance Fas expression. The increased Fas expression in the regions containing many dead or dying cells2 suggests a link between expression of Fas and SMC apoptosis within the lesions.
The colocalization of Fas+ SMCs with activated T cells in the plaques may thus have pathophysiologic significance. Human atherosclerotic plaques contain both CD4+ and CD8+ T cells.9 Activated CD8+ T cells can produce FasL and can induce apoptosis of target cells. Although a soluble form of FasL may exist, the membrane-bound form of FasL seems to play a major role in the Fas-mediated killing. Induction of apoptosis in SMCs may involve direct contact between the T cells expressing FasL and SMCs with Fas augmented by locally produced cytokines. Such a mechanism could explain the increased number of TUNEL+ cells in the areas where T cells and macrophages accumulate. However, the expression and function of FasL in the atherosclerotic lesions remain to be investigated.
Our in vitro experiments support the possibility that cytokines
produced by T cells and macrophages enhance expression of Fas
in human vascular SMCs. The observation that cytokines augment
expression of Fas in human vascular SMCs agrees well with recent
findings obtained by other laboratories demonstrating that
cytokines enhance expression of Fas in a broad variety of cell
types.22–24 Cytokines can act
synergistically in this regard. The presence of TNF and IL-1 augment
IFN-–induced Fas expression and Fas-mediated killing. The mechanism
of this synergy remains unclear. Fukuo et al36
recently reported that ·NO released from rat vascular SMCs stimulated
with IL-1 promotes expression of Fas in a cGMP-independent fashion.
Indeed, the proinflammatory cytokines studied here synergize to
induce synthesis of ·NO in rodent SMCs.37,38 In
contrast to rodent SMCs, however, human SMCs produce little ·NO in
response to the cytokines studied here.16
It is therefore unlikely that ·NO mediates the expression of Fas in
the cytokine-stimulated human SMCs.
Fas expression does not cause apoptosis per se because many cell types in normal tissues, including arterial tissue with few dead cells, can constitutively produce Fas. We observed that in culture, human SMCs spontaneously express considerable levels of Fas but do not undergo apoptosis even after incubating with the activating antibody CH-11. We found that cultured SMCs actually resist apoptosis induced by Fas ligation even in the absence of serum-growth factors. In view of the basal Fas expression in plaque cells and in cultured SMCs, cytokine pretreatment may trigger apoptosis not only by augmenting expression of Fas but also affecting downstream events in the death-signaling pathway. Moreover, activation of the Fas death-signaling pathway requires ligation of Fas with FasL or anti-Fas antibody.39 These cytokines may have effects on production of FasL or related proteins. SMCs freshly isolated from normal aorta and from atherosclerotic plaques show different apoptotic responses. Bennett et al18 recently reported an increased sensitivity of human SMCs from advanced atherosclerotic plaques to apoptotic attack by serum starvation. However, it remains unclear whether this is mediated by the Fas/FasL death-signaling pathway.
Recently, several laboratories have identified a group of intracellular proteolytic enzymes in the downstream steps of the apoptotic cascade triggered by activation of Fas.25 Sequence comparison and proteolytic function assay suggest that these proteases belong to the ICE gene family and share similar but not identical substrate specificity, eg, cleavage of peptides at Asp-X bonds.40 Inhibition of ICE and its related enzymes with the ICE inhibitor such as the baculovirus protein p35 41 and the Cowpox virus protein crmA42,43 can block apoptosis triggered by activation of Fas. Expression and function of the ICE family may possibly function in atherosclerotic lesions. We recently observed that human atherosclerotic lesions express both mRNA and protein of ICE.2 Double staining with a combination of immunohistochemistry and TUNEL shows colocalization of the ICE antigen with TUNEL+ SMCs in human atherosclerotic lesions,2 suggesting association of ICE with apoptosis of SMCs induced by activation of the Fas pathway. We have also found the expression of other isoforms of ICE such as CPP32 and ICH-1 L in human atherosclerotic plaques (Geng et al, unpublished observations). Thus, the ICE family may contribute to apoptosis of Fas-expressing SMCs.
Although we show evidence in this article that the Fas/FasL system participates in apoptosis of vascular SMCs, other death pathways may operate in atherosclerotic lesions. For example, cells in the plaques may die by oncosis. The observation that TUNEL+ cells localize in the region containing many lipid-laden foamy macrophages and oxidized lipids points to the cytotoxicity of activated macrophages and accumulation of mediators that may interact with Fas and/or cytokines in induction of cell death.
The pathologic and clinical significance of apoptosis in atherosclerosis remains controversial. As a process of normal or physiologic cell death, apoptosis may counteract mitosis, limit cell accumulation, and consequently inhibit intimal thickening, a key event in the pathogenesis of atherosclerosis and restenosis. Cells undergoing apoptosis can maintain intact cell membrane and therefore cause little inflammatory reaction. Apoptosis may prove adaptive by elimination of unwanted or harmful cells without insulting the vessel wall.
However, immunogenic stimuli derived from many atherogenic substances including the products of microorganisms and modified lipoproteins may activate infiltrating immune cells that, in turn, trigger apoptosis of vascular cells via the Fas/FasL pathway. The cytokine-induced, Fas-mediated apoptosis at high levels may lead to accelerated death of SMCs, the most abundant cellular component of arteries. Such SMC death, together with increased release of matrix-digesting enzymes and accumulation of lipids, may substantially weaken the vessel wall and promote rupture of atherosclerotic plaques, a major cause of acute coronary syndromes. In addition, if not removed, apoptotic cells may accumulate among lipids and matrix. Finally, increased accumulation of calcium in cells undergoing apoptosis can promote plaque calcification, a common feature of advanced atherosclerotic plaques. Therefore, understanding of the molecular mechanisms for induction of apoptosis and subsequent biochemical changes in vascular cells may provide a link between immune activation and the evolution of this vascular disease.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by National Institutes of Health grant HL-34636. We thank Dr F.J. Schoen of Brigham and Women's Hospital, Harvard Medical School, for providing paraffin sections of human atherosclerotic plaques.
Received August 30, 1996; accepted January 13, 1997.
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Wyllie AH, Kerr JFR, Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol. 1980;68:251-306.[Medline] [Order article via Infotrieve]
2. Geng Y-J, Libby P. Evidence for apoptosis in advanced human atheroma: colocalization with interleukin-1b converting enzyme. Am J Pathol. 1995;147:251-266.[Abstract]
3. Han DKM, Haudenshild CC, Hong MK, Thurmin A, Liau G. Evidence for a possible role of apoptosis in atherogenesis. Am J Pathol. 1995;147:267-277.[Abstract]
4.
Isner JM, Kearney M, Bortman S, Passeri J.
Apoptosis in human atherosclerosis and
restenosis. Circulation. 1995;91:2703-2711.
5. Kockx MM, Muhring J, Bortier M, De Meyer GR, Jacob W. Biotin- or digoxigenin-conjugated nucleotides bind to matrix vesicle in atherosclerotic plaques. Am J Pathol. 1996;148:1771-1777.[Abstract]
6. Bjorkerud S, Bjorkerud B. Apoptosis is abundant in human atherosclerotic lesions, especially in inflammatory cells (macrophages and T cells), and may contribute to the accumulation of gruel and plaque instability. Am J Pathol. 1996;149:367-380.[Abstract]
7. Kockx MM, De Meyer GR, Muhring J, Bult H, Bultinck J, Herman AG. Distribution of cell replication and apoptosis in atherosclerotic plaques of cholesterol-fed rabbits. Atherosclerosis. 1996;120:115-124.[Medline] [Order article via Infotrieve]
8. Bochaton-Piallat M-L, Gabbiani F, Redard M, Desmouliere A, Gabbiani G. Apoptosis participates in cellularity regulation during rat aortic intimal thickening. Am J Pathol. 1995;146:1-6.[Medline] [Order article via Infotrieve]
9. Libby P, Hansson GK. Involvement of the immune system in human atherogenesis: current knowledge and unanswered questions. Lab Invest. 1991;64:5-15.[Medline] [Order article via Infotrieve]
10.
Geng Y-J, Holm J, Nygren S, Bruzelius M, Stemme S,
Hansson G. Expression of the macrophage scavenger receptor in
atheroma. Relationship to immune activation and the T cell
cytokine, interferon-gamma. Arterioscler Thromb Vasc
Biol. 1995;15:1995-2002.
11. Libby P, Clinton SK. Cytokines as mediators of vascular pathology. Nouv Rev Fr Hematol. 1992;34(suppl):S47-S53.
12.
Hansson GK, Jonasson L, Holm J, Clowes MM, Clowes AW.
Interferon-gamma regulates vascular smooth muscle cell proliferation
and Ia antigen expression in vivo and in vitro. Circ Res. 1988;63:712-719.
13.
Hansson GK, Hellstrand M, Rymo L, Rubbia L, Gabbiani G.
Interferon-gamma inhibits proliferation and expression of
differentiation-specific alpha-smooth muscle actin in
arterial smooth muscle cells. J Exp Med. 1989;170:1595-1608.
14. Warner SJC, Libby P. Human smooth muscle cells. Target for and source of tumor necrosis factor. J Immunol. 1989;142:100-109.[Abstract]
15. Libby P, Warner SJC, Friedman GB. Interleukin-1: a mitogen for human vascular smooth muscle cells that induces the release of growth-inhibitory prostanoids. J Clin Invest. 1988;88:487-498.
16.
Geng Y-J, Wu Q, Muszynski M, Hansson G, Libby P.
Apoptosis of vascular smooth muscle cells induced by in vitro
stimulation with interferon-gamma, tumor necrosis factor-alpha, and
interleukin-1beta. Arterioscler Thromb Vasc Biol. 1996;16:19-27.
17.
Bennett MR, Evan GI, Newby AC. Deregulated expression
of the c-myc oncogene abolishes inhibition of proliferation of rat
vascular smooth muscle cells by serum reduction, interferon-g, heparin,
and cyclic nucleotide analogues and induces
apoptosis. Circ Res. 1994;74:525-536.
18. Bennett MR, Evan GI, Schwartz SM. Apoptosis of human vascular smooth muscle cells derived from normal vessels and coronary atherosclerotic plaques. J Clin Invest. 1995;95:2266-2274.
19.
Nagata S, Golstein P. The Fas death factor.
Science. 1995;267:1449-1456.
20. Watanabe D, Suda T, Hashimoto H, Nagata S. Constitutive activation of the Fas ligand gene in mouse lymphoproliferative disorders. EMBO J. 1995;14:12-18.[Medline] [Order article via Infotrieve]
21. Watanabe-Fukunaga R, Brannan CI, Itoh N, Yonehara S, Copeland NG, Jenkins NA, Nagata S. The cDNA structure, expression, and chromosomal assignment of the mouse Fas antigen. J Immunol. 1992;148:1274-1279.[Abstract]
22.
Maciejewski J, Selleri C, Anderson S, Young NS. Fas
antigen expression on CD34+ human marrow cells is induced by interferon
gamma and tumor necrosis factor alpha and potentiates
cytokine-mediated hematopoietic suppression in vitro.
Blood. 1995;85:3183-3190.
23. Takahashi H, Kobayashi H, Hashimoto Y, Matsuo S, Iizuka H. Interferon-gamma-dependent stimulation of Fas antigen in SV40-transformed human keratinocytes: modulation of the apoptotic process by protein kinase C. J Invest Dermatol. 1995;105:810-815.[Medline] [Order article via Infotrieve]
24. Weller M, Frei K, Groscurth P, Krammer PH, Yonekawa Y, Fontana A. Anti-Fas/APO-1 antibody-mediated apoptosis of cultured human glioma cells. Induction and modulation of sensitivity by cytokines. J Clin Invest. 1994;94:954-964.
25. Fraser A, Evan G. A license to kill. Cell. 1996;85:781-784.[Medline] [Order article via Infotrieve]
26. Suda T, Takahashi T, Golstein P, Nagata S. Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family. Cell. 1993;75:1169-1178.[Medline] [Order article via Infotrieve]
27. Suda T, Okazaki T, Naito Y, Yokota T, Arai N, Ozaki S, Nakao K, Nagata S. Expression of the Fas ligand in cells of T cell lineage. J Immunol. 1995;154:3806-3813.[Abstract]
28. Libby P, O'Brien KV. Culture of quiescent vascular smooth muscle cells in a defined serum-free medium. J Cell Physiol. 1983;115:217-233.[Medline] [Order article via Infotrieve]
29.
Gavrieli Y, Sherman Y, Ben SS. Identification of
programmed cell death in situ via specific labeling of nuclear DNA
fragmentation. J Cell Biol. 1992;119:493-501.
30. Wijsman JH, Jonker RR, Keijzer R, van de Velde CJ, Cornelisse CJ, van Dierendonck JH. A new method to detect apoptosis in paraffin sections: in situ end-labeling of fragmented DNA. J Histochem Cytochem. 1993;41:7-12.[Abstract]
31. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-160.[Medline] [Order article via Infotrieve]
32. Itoh N, Yonehara S, Ishii A, Yonehara M, Mizushima S, Sameshima M, Hase A, Seto Y, Nagata S. The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell. 1991;66:233-243.[Medline] [Order article via Infotrieve]
33. Weis M, Schlegel J, Kass GE, Holmstrom TH, Peters I, Eriksson J, Orrenius S, Chow SC. Cellular events in Fas/APO-1-mediated apoptosis in JURKAT T lymphocytes. Exp Cell Res. 1995;219:699-708.[Medline] [Order article via Infotrieve]
34.
Weller M, Malipiero U, Rensing-Ehl A, Barr PJ, Fontana
A. Fas/APO-1 gene transfer for human malignant glioma. Cancer
Res. 1995;55:2936-2944.
35. Hansson GK, Holm J, Jonasson L. Detection of activated T lymphocytes in the human atherosclerotic plaque. Am J Path. 1989;135:169-175.[Abstract]
36. Fukuo K, Hata S, Suhara T, Nakahashi T, Shinto Y, Tsujimoto Y, Morimoto S, Ogihara T. Nitric oxide induces upregulation of Fas and apoptosis in vascular smooth muscle. Hypertension. 1996;27(pt 2):823-826.
37.
Geng Y, Hansson GK, Holme E. Interferon-gamma and tumor
necrosis factor synergize to induce nitric oxide production and
inhibit mitochondrial respiration in vascular smooth muscle cells.
Circ Res. 1992;71:1268-1276.
38. Geng YJ, Petersson AS, Wennmalm A, Hansson GK. Cytokine-induced expression of nitric oxide synthase results in nitrosylation of heme and nonheme iron proteins in vascular smooth muscle cells. Exp Cell Res. 1994;214:418-428.[Medline] [Order article via Infotrieve]
39. Nagata S. Fas and Fas ligand: a death factor and its receptor. Adv Immunol. 1994;57:129-144.[Medline] [Order article via Infotrieve]
40.
Steller H. Mechanisms and genes of cellular suicide.
Science. 1995;267:1445-1449.
41.
Beidler DR, Tewari M, Friesen PD, Poirier G, Dixit VM.
The baculovirus p35 protein inhibits Fas- and tumor necrosis
factor-induced apoptosis. J Biol Chem. 1995;270:16526-16528.
42.
Tewari M, Dixit VM. Fas- and tumor necrosis
factor-induced apoptosis is inhibited by the poxvirus crmA gene
product. J Biol Chem. 1995;270:3255-3260.
43.
Tewari M, Beidler DR, Dixit VM. CrmA-inhibitable
cleavage of the 70-kDa protein component of the U1 small nuclear
ribonucleoprotein during Fas- and tumor necrosis factor-induced
apoptosis. J Biol Chem. 1995;270:18738-18741.
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|
|
|
|
|
|
R. Kraemer Reduced Apoptosis and Increased Lesion Development in the Flow-Restricted Carotid Artery of p75NTR-Null Mutant Mice Circ. Res., September 20, 2002; 91(6): 494 - 500. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
|
|
T. Aoyama, G. Takemura, R. Maruyama, K.-i. Kosai, T. Takahashi, M. Koda, K. Hayakawa, Y. Kawase, S. Minatoguchi, and H. Fujiwara Molecular mechanisms of non-apoptosis by Fas stimulation alone versus apoptosis with an additional actinomycin D in cultured cardiomyocytes Cardiovasc Res, September 1, 2002; 55(4): 787 - 798. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. J. Pinderski, M. P. Fischbein, G. Subbanagounder, M. C. Fishbein, N. Kubo, H. Cheroutre, L. K. Curtiss, J. A. Berliner, and W. A. Boisvert Overexpression of Interleukin-10 by Activated T Lymphocytes Inhibits Atherosclerosis in LDL Receptor-Deficient Mice by Altering Lymphocyte and Macrophage Phenotypes Circ. Res., May 31, 2002; 90(10): 1064 - 1071. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Masse, M.-J. Hebert, S. Troyanov, N. Vigneault, I. Sirois, and F. Madore Soluble Fas is a marker of peripheral arterial occlusive disease in haemodialysis patients Nephrol. Dial. Transplant., March 1, 2002; 17(3): 485 - 491. [Abstract] [Full Text] [PDF]
|
|
|
|
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 T. Suhara, H.-S. Kim, L. A. Kirshenbaum, and K. Walsh Suppression of Akt Signaling Induces Fas Ligand Expression: Involvement of Caspase and Jun Kinase Activation in Akt-Mediated Fas Ligand Regulation Mol. Cell. Biol., January 15, 2002; 22(2): 680 - 691. [Abstract] [Full Text] [PDF]
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P. N. Seshiah, D. J. Kereiakes, S. S. Vasudevan, N. Lopes, B. Y. Su, N. A. Flavahan, and P. J. Goldschmidt-Clermont Activated Monocytes Induce Smooth Muscle Cell Death: Role of Macrophage Colony-Stimulating Factor and Cell Contact Circulation, January 15, 2002; 105(2): 174 - 180. [Abstract] [Full Text] [PDF]
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G. K. Hansson Immune Mechanisms in Atherosclerosis Arterioscler Thromb Vasc Biol, December 1, 2001; 21(12): 1876 - 1890. [Abstract] [Full Text] [PDF]
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M. Sata, S. Sugiura, M. Yoshizumi, Y. Ouchi, Y. Hirata, and R. Nagai Acute and Chronic Smooth Muscle Cell Apoptosis After Mechanical Vascular Injury Can Occur Independently of the Fas-Death Pathway Arterioscler Thromb Vasc Biol, November 1, 2001; 21(11): 1733 - 1737. [Abstract] [Full Text] [PDF]
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J. J. Boyle, D. E. Bowyer, P. L. Weissberg, and M. R. Bennett Human Blood-Derived Macrophages Induce Apoptosis in Human Plaque-Derived Vascular Smooth Muscle Cells by Fas-Ligand/Fas Interactions Arterioscler Thromb Vasc Biol, September 1, 2001; 21(9): 1402 - 1407. [Abstract] [Full Text] [PDF]
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A. J. Belanger, A. Scaria, H. Lu, J. A. Sullivan, S. H. Cheng, R. J. Gregory, and C. Jiang Fas ligand/Fas-mediated apoptosis in human coronary artery smooth muscle cells: therapeutic implications of fratricidal mode of action Cardiovasc Res, September 1, 2001; 51(4): 749 - 761. [Abstract] [Full Text] [PDF]
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P. Libby Current Concepts of the Pathogenesis of the Acute Coronary Syndromes Circulation, July 17, 2001; 104(3): 365 - 372. [Full Text] [PDF]
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 T.-S. Lee and L.-Y. Chau Fas/Fas ligand-mediated death pathway is involved in oxLDL-induced apoptosis in vascular smooth muscle cells Am J Physiol Cell Physiol, March 1, 2001; 280(3): C709 - C718. [Abstract] [Full Text] [PDF]
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Y. Okura, M. Brink, H. Itabe, K. J. Scheidegger, A. Kalangos, and P. Delafontaine Oxidized Low-Density Lipoprotein Is Associated With Apoptosis of Vascular Smooth Muscle Cells in Human Atherosclerotic Plaques Circulation, November 28, 2000; 102(22): 2680 - 2686. [Abstract] [Full Text] [PDF]
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 C. NAPOLI, O. QUEHENBERGER, F. DE NIGRIS, P. ABETE, C. K. GLASS, and W. PALINSKI Mildly oxidized low density lipoprotein activates multiple apoptotic signaling pathways in human coronary cells FASEB J, October 1, 2000; 14(13): 1996 - 2007. [Abstract] [Full Text]
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S. Wang, P. Bray, T. McCaffrey, K. March, B. L. Hempstead, and R. Kraemer p75NTR Mediates Neurotrophin-Induced Apoptosis of Vascular Smooth Muscle Cells Am. J. Pathol., October 1, 2000; 157(4): 1247 - 1258. [Abstract] [Full Text] [PDF]
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F. D. Kolodgie, J. Narula, A. P. Burke, N. Haider, A. Farb, Y. Hui-Liang, J. Smialek, and R. Virmani Localization of Apoptotic Macrophages at the Site of Plaque Rupture in Sudden Coronary Death Am. J. Pathol., October 1, 2000; 157(4): 1259 - 1268. [Abstract] [Full Text] [PDF]
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T. Imanishi, C. E. Murry, H. Reinecke, T. Hano, I. Nishio, W.C. Liles, L. Hofsta, K. Kim, K. D. O'Brien, S. M. Schwartz, et al. Cellular FLIP is expressed in cardiomyocytes and down-regulated in TUNEL-positive grafted cardiac tissues Cardiovasc Res, October 1, 2000; 48(1): 101 - 110. [Abstract] [Full Text] [PDF]
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K. Walsh, R. C. Smith, and H.-S. Kim Vascular Cell Apoptosis in Remodeling, Restenosis, and Plaque Rupture Circ. Res., August 4, 2000; 87(3): 184 - 188. [Full Text] [PDF]
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G. H. Gibbons and M. J. Pollman Death Receptors, Intimal Disease, and Gene Therapy : Are Therapies That Modify Cell Fate Moving too Fas? Circ. Res., May 26, 2000; 86(10): 1009 - 1012. [Full Text] [PDF]
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S.-W. Chan, L. Hegyi, S. Scott, N. R. B. Cary, P. L. Weissberg, and M. R. Bennett Sensitivity to Fas-Mediated Apoptosis Is Determined Below Receptor Level in Human Vascular Smooth Muscle Cells Circ. Res., May 26, 2000; 86(10): 1038 - 1046. [Abstract] [Full Text] [PDF]
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R. Virmani, F. D. Kolodgie, A. P. Burke, A. Farb, and S. M. Schwartz Lessons From Sudden Coronary Death : A Comprehensive Morphological Classification Scheme for Atherosclerotic Lesions Arterioscler Thromb Vasc Biol, May 1, 2000; 20(5): 1262 - 1275. [Full Text] [PDF]
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H.-S. Kim, K.-K. Hwang, J.-W. Seo, S.-Y. Kim, B.-H. Oh, M.-M. Lee, and Y.-B. Park Apoptosis and Regulation of Bax and Bcl-X Proteins During Human Neonatal Vascular Remodeling Arterioscler Thromb Vasc Biol, April 1, 2000; 20(4): 957 - 963. [Abstract] [Full Text] [PDF]
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 K. J. Hamann, J. E. Vieira, A. J. Halayko, D. Dorscheid, S. R. White, S. M. Forsythe, B. Camoretti-Mercado, K. F. Rabe, and J. Solway Fas cross-linking induces apoptosis in human airway smooth muscle cells Am J Physiol Lung Cell Mol Physiol, March 1, 2000; 278(3): L618 - L624. [Abstract] [Full Text] [PDF]
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D. B. Schneider, G. Vassalli, S. Wen, R. M. Driscoll, A. B. Sassani, M. B. DeYoung, R. Linnemann, R. Virmani, and D. A. Dichek Expression of Fas Ligand in Arteries of Hypercholesterolemic Rabbits Accelerates Atherosclerotic Lesion Formation Arterioscler Thromb Vasc Biol, February 1, 2000; 20(2): 298 - 308. [Abstract] [Full Text] [PDF]
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M. Sata, T. Suhara, and K. Walsh Vascular Endothelial Cells and Smooth Muscle Cells Differ in Expression of Fas and Fas Ligand and in Sensitivity to Fas Ligand-Induced Cell Death : Implications for Vascular Disease and Therapy Arterioscler Thromb Vasc Biol, February 1, 2000; 20(2): 309 - 316. [Abstract] [Full Text] [PDF]
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M. M Kockx and A. G Herman Apoptosis in atherosclerosis: beneficial or detrimental? Cardiovasc Res, February 1, 2000; 45(3): 736 - 746. [Abstract] [Full Text] [PDF]
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N. J. McCarthy and M. Bennett The regulation of vascular smooth muscle cell apoptosis Cardiovasc Res, February 1, 2000; 45(3): 747 - 755. [Abstract] [Full Text] [PDF]
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K. Walsh and J. M. Isner Apoptosis in inflammatory-fibroproliferative disorders of the vessel wall Cardiovasc Res, February 1, 2000; 45(3): 756 - 765. [Abstract] [Full Text] [PDF]
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T. Imanishi, J. McBride, Q. Ho, K. D. O’Brien, S. M. Schwartz, and D. K. M. Han Expression of Cellular FLICE-Inhibitory Protein in Human Coronary Arteries and in a Rat Vascular Injury Model Am. J. Pathol., January 1, 2000; 156(1): 125 - 137. [Abstract] [Full Text] [PDF]
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P. Aukrust, F. Muller, T. Ueland, T. Berget, E. Aaser, A. Brunsvig, N. O. Solum, K. Forfang, S. S. Froland, and L. Gullestad Enhanced Levels of Soluble and Membrane-Bound CD40 Ligand in Patients With Unstable Angina : Possible Reflection of T Lymphocyte and Platelet Involvement in the Pathogenesis of Acute Coronary Syndromes Circulation, August 10, 1999; 100(6): 614 - 620. [Abstract] [Full Text] [PDF]
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S. N. Orlov, N. Thorin-Trescases, S. V. Kotelevtsev, J. Tremblay, and P. Hamet Inversion of the Intracellular Na+/K+ Ratio Blocks Apoptosis in Vascular Smooth Muscle at a Site Upstream of Caspase-3 J. Biol. Chem., June 4, 1999; 274(23): 16545 - 16552. [Abstract] [Full Text] [PDF]
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M. R Bennett Apoptosis of vascular smooth muscle cells in vascular remodelling and atherosclerotic plaque rupture Cardiovasc Res, February 1, 1999; 41(2): 361 - 368. [Abstract] [Full Text] [PDF]
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E. Lutgens, E. D. de Muinck, P. J.E.H.M. Kitslaar, J. H.M. Tordoir, H. J.J. Wellens, and M. J.A.P. Daemen Biphasic pattern of cell turnover characterizes the progression from fatty streaks to ruptured human atherosclerotic plaques Cardiovasc Res, February 1, 1999; 41(2): 473 - 479. [Abstract] [Full Text] [PDF]
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E. L. Henderson, Y.-J. Geng, G. K. Sukhova, A. D. Whittemore, J. Knox, and P. Libby Death of Smooth Muscle Cells and Expression of Mediators of Apoptosis by T Lymphocytes in Human Abdominal Aortic Aneurysms Circulation, January 12, 1999; 99(1): 96 - 104. [Abstract] [Full Text] [PDF]
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M. M. Kockx Apoptosis in the Atherosclerotic Plaque : Quantitative and Qualitative Aspects Arterioscler Thromb Vasc Biol, October 1, 1998; 18(10): 1519 - 1522. [Abstract] [Full Text] [PDF]
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M. M. Kockx, G. R. Y. De Meyer, N. Buyssens, M. W. M. Knaapen, H. Bult, and A. G. Herman Cell Composition, Replication, and Apoptosis in Atherosclerotic Plaques After 6 Months of Cholesterol Withdrawal Circ. Res., August 24, 1998; 83(4): 378 - 387. [Abstract] [Full Text] [PDF]
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A. Haunstetter and S. Izumo Apoptosis : Basic Mechanisms and Implications for Cardiovascular Disease Circ. Res., June 15, 1998; 82(11): 1111 - 1129. [Full Text] [PDF]
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M. M. Kavurma, F. S. Santiago, E. Bonfoco, and L. M. Khachigian Sp1 Phosphorylation Regulates Apoptosis via Extracellular FasL-Fas Engagement J. Biol. Chem., February 9, 2001; 276(7): 4964 - 4971. [Abstract] [Full Text] [PDF]
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J. H. von der Thusen, B. J.M. van Vlijmen, R. C. Hoeben, M. M. Kockx, L.M. Havekes, T. J.C. van Berkel, and E. A.L. Biessen Induction of Atherosclerotic Plaque Rupture in Apolipoprotein E-/- Mice After Adenovirus-Mediated Transfer of p53 Circulation, April 30, 2002; 105(17): 2064 - 2070. [Abstract] [Full Text] [PDF]
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